Synthetic gene encoding rhesus monkey carcinoembryonic antigen and uses thereof

ABSTRACT

Synthetic polynucleotides encoding rhesus monkey carcinoembryonic antigen (CEA) are provided, the synthetic polynucleotides being condon-optimized for expression in a human cellular environment. The gene encoding CEA is commonly associated with the development of human carcinomas. The present invention provides compositions and methods to elicit or enhance immunity to the protein product expressed by the CEA tumor-associated antigen, wherein aberrant CEA expression is associated with a carcinoma or its development. This invention specifically provides adenovial vector and plasmid constructs carrying codon-optimed rhesus monkey CEA and discloses their use in vaccines and pharmaceutical compositions for preventing and treating cancer.

FIELD OF THE INVENTION

The present invention relates generally to the therapy of cancer. Morespecifically, the present invention relates to synthetic polynucleotidesencoding the rhesus monkey homologue of the human tumor associatedpolypeptide carcinoembryonic antigen, herein designated rhCEAopt,wherein the polynucleotides are codon-optimized for expression in ahuman cellular environment. The present invention also providesrecombinant vectors and hosts comprising said synthetic polynucleotides.This invention also relates to adenoviral vector and plasmid constructscarrying rhCEAopt and to their use in vaccines and pharmaceuticalcompositions for preventing and treating cancer.

BACKGROUND OF THE INVENTION

The immunoglobulin superfamily (IgSF) consists of numerous genes thatcode for functionally diverse proteins. One important function of IgSFproteins is intercellular adhesion. IgSF proteins contain at least oneIg-related domain that is important for maintaining properintermolecular binding interactions. Because such interactions arenecessary to the diverse biological functions of the IgSF members,disruption or aberrant expression of many IgSF adhesion molecules hasbeen correlated with human disease.

The carcinoembryonic antigen (CEA) belongs to a subfamily of the IgSFconsisting of cell surface glycoproteins. Members of the CEA subfamilyare known as CEA-related cell adhesion molecules (CEACAMs). The genethat encodes the CEA protein is often referred to as CEACAM5.Functionally, CEACAMs have been shown to act as both homotypic andheterotypic intercellular adhesion molecules (Benchimol et al., Cell57:327-334 (1989)). In addition to cell adhesion, CEA inhibits celldeath resulting from detachment of cells from the extracellular matrixand contributes to cellular transformation associated with certainproto-oncogenes such as Bcl2 and C-Myc (see Berinstein, J. Clin Oncol.20(8): 2197-2207 (2002)).

Sequences coding for wild-type human CEA have been cloned andcharacterized (U.S. Pat. No. 5,274,087;U.S. Pat. No. 5,571,710; and U.S.Pat. No. 5,843,761. See also Beauchemin et al., Mol. Cell. Biol.7:3221-3230 (1987); Zimmerman et al., Proc. Natl. Acad. Sci. USA84:920-924 (1987); Thompson et al. Proc. Natl. Acad. Sci. USA84(9):2965-69 (1987)).

Expression of CEA is normally detected during fetal development and inadult colonic mucosa. Overexpression of CEA is commonly associated withvarious malignancies. Such overexpression was first detected in humancolon tumors over thirty years ago (Gold and Freedman, J. Exp. Med.121:439-462 (1965)), and has since been found in nearly all colorectaltumors. Additionally, CEA overexpression is detectable in a highpercentage of adenocarcinomas of the pancreas, breast and lung. Becauseof the prevalence of CEA expression in these tumor types, CEA is widelyused clinically in the management and prognosis of these cancers.

The correlation between CEA expression and metastatic growth has led toits identification as a target for molecular and immunologicalintervention for colorectal cancer treatment. One therapeutic approachtargeting CEA is the use of anti-CEA antibodies (see Chester et al.,Cancer Chemother. Pharmacol. 46 (Suppl): S8-S12 (2000)). Anotherapproach is the activation of the immune system to attack CEA-expressingtumors using CEA-based vaccines (for review, see Berinstein, supra).However, because CEA is a normal self-component that is overexpressed incancer cells, specific immunotherapy targeting CEA must overcomeself-tolerance.

The development and commercialization of many vaccines have beenhindered by difficulties associated with obtaining high expressionlevels of exogenous genes in successfully transformed host organisms.Therefore, despite the identification of the wild-type nucleotidesequences encoding CEA proteins described above, it would be highlydesirable to develop a readily renewable source of CEA protein thatutilizes CEA-encoding nucleotide sequences that are optimized forexpression in the intended host cell, said source allowing for thedevelopment of a cancer vaccine which is efficacious and not hindered byself-tolerance.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods to elicit orenhance immunity to the protein products expressed by CEA genes, whichhave been associated with numerous adenocarcinomas, including colorectalcarcinomas. Specifically, the present invention provides polynucleotidesencoding rhesus monkey CEA protein, wherein said polynucleotides arecodon-optimized for high level expression in a human cell. The presentinvention further provides adenoviral and plasmid-based vectorscomprising the synthetic polynucleotides and discloses use of saidvectors in immunogenic compositions and vaccines for the preventionand/or treatment of CEA-associated cancer.

The present invention also relates to synthetic nucleic acid molecules(polynucleotides) comprising a sequence of nucleotides that encoderhesus monkey carcinoembryonic antigen (hereinafter rhCEA) as set forthin SEQ ID NO:2 or SEQ ID NO:3, wherein the synthetic nucleic acidmolecules are codon-optimized for high-level expression in a human cell(hereinafter rhCEAopt). The nucleic acid molecules disclosed herein maybe transfected into a host cell of choice wherein the recombinant hostcell provides a source for substantial levels of an expressed functionalrhCEA protein (SEQ ID NOs:2 and 3).

The present invention further relates to a synthetic nucleic acidmolecule which encodes mRNA that expresses a rhesus monkey CEA protein;this DNA molecule comprising the nucleotide sequence disclosed herein asSEQ ID NO:1. A preferred aspect of this portion of the present inventionis disclosed in FIG. 1, which shows a DNA molecule (SEQ ID NO:1) thatencodes a rhCEA protein (SEQ ID NO:2). The preferred nucleic acidmolecule of the present invention is codon-optimized for high-levelexpression in a human cell.

The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which containthe nucleic acid molecules disclosed throughout this specification.

The present invention further relates to a process for expressing acodon-optimized rhesus monkey CEA protein in a recombinant host cell,comprising: (a) introducing a vector comprising a nucleic acid moleculethat encodes rhesus monkey CEA protein into a suitable host cell,wherein the nucleic acid molecule is codon-optimized for optimalexpression in the host cell; and, (b) culturing the host cell underconditions which allow expression of said codon-optimized rhesus monkeyprotein.

Another aspect of this invention is a method of preventing or treatingcancer comprising administering to a mammal a vaccine vector comprisinga synthetic nucleic acid molecule, the synthetic nucleic acid moleculecomprising a sequence of nucleotides that encodes a rhesus monkeycarcinoembryonic antigen (rhCEA) protein as set forth in SEQ ID NO:2 oras set forth in SEQ ID NO:3, wherein the synthetic nucleic acid moleculeis codon-optimized for high level expression in a human cell.

The present invention further relates to an adenovirus vaccine vectorcomprising an adenoviral genome with a deletion in the E1 region, and aninsert in the E1 region, wherein the insert comprises an expressioncassette comprising: (a) a codon-optimized polynucleotide encoding arhesus monkey CEA protein; and (b) a promoter operably linked to thepolynucleotide.

The present invention also relates to a vaccine plasmid comprising aplasmid portion and an expression cassette portion, the expressioncassette portion comprising: (a) a synthetic polynucleotide encoding arhesus monkey CEA protein, wherein the synthetic polynucleotide iscodon-optimized for optimal expression in a human cell; and (b) apromoter operably linked to the polynucleotide.

Another aspect of the present invention is a method of protecting amammal from cancer or treating a mammal suffering from CEA-associatedcancer comprising: (a) introducing into the mammal a first vectorcomprising: i) a codon-optimized polynucleotide encoding a rhesus monkeyCEA protein; and ii) a promoter operably linked to the polynucleotide;(b) allowing a predetermined amount of time to pass; and (c) introducinginto the mammal a second vector comprising: i) a codon-optimizedpolynucleotide encoding a rhesus monkey CEA protein; and ii) a promoteroperably linked to the polynucleotide.

As used throughout the specification and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise.

As used throughout the specification and appended claims, the followingdefinitions and abbreviations apply:

The term “promoter” refers to a recognition site on a DNA strand towhich the RNA polymerase binds. The promoter forms an initiation complexwith RNA polymerase to initiate and drive transcriptional activity. Thecomplex can be modified by activating sequences termed “enhancers” orinhibiting sequences termed “silencers”.

The terrn “cassette” refers to the sequence of the present inventionthat contains the nucleic acid sequence which is to be expressed. Thecassette is similar in concept to a cassette tape; each cassette has itsown sequence. Thus by interchanging the cassette, the vector willexpress a different sequence. Because of the restriction sites at the 5′and 3′ ends, the cassette can be easily inserted, removed or replacedwith another cassette.

The term “vector” refers to some means by which DNA fragments can beintroduced into a host organism or host tissue. There are various typesof vectors including plasmid, virus (including adenovirus),bacteriophages and cosmids.

The term “first generation,” as used in reference to adenoviral vectors,describes said adenoviral vectors that are replication-defective. Firstgeneration adenovirus vectors typically have a deleted or inactivated E1gene region, and preferably have a deleted or inactivated E3 generegion.

The designation “pV1J-rhCEAopt” refers to a plasmid construct, disclosedherein, comprising the human CMV immediate-early (IE) promoter withintron A, a full-length codon-optimized human CEA gene, bovine growthhormone-derived polyadenylation and transcriptional terminationsequences, and a minimal pUC backbone (see EXAMPLE 2). The designation“pV1J-rhCEA” refers to a construct as described above, except theconstruct comprises a wild-type rhesus monkey CEA gene instead of acodon-optimized rhesus monkey CEA gene.

The designations “MRKAd5/rhCEAopt” and “MRKAd5/rhCEA” refer to twoconstructs, disclosed herein, which comprise an Ad5 adenoviral genomedeleted of the E1 and E3 regions. In the “MRKAd5/rhCEAopt” construct,the E1 region is replaced by a codon-optimized rhesus monkey CEA gene inan E1 parallel orientation under the control of a human CMV promoterwithout intron A, followed by a bovine growth hormone polyadenylationsignal. The “MRKAd5rhCEA” construct is essentially as described above,except the E1 region of the AdS genome is replaced with a wild-typerhesus monkey CEA sequence (see EXAMPLE 2).

The term “effective amount” means sufficient vaccine composition isintroduced to produce the adequate levels of the polypeptide, so that animmune response results. One skilled in the art recognizes that thislevel may vary.

A “conservative amino acid substitution” refers to the replacement ofone amino acid residue by another, chemically similar, amino acidresidue. Examples of such conservative substitutions are: substitutionof one hydrophobic residue (isoleucine, leucine, valine, or methionine)for another; substitution of one polar residue for another polar residueof the same charge (e.g., arginine for lysine; glutainic acid foraspartic acid).

“rhCEA” and “rbCEAopt” refer to a rhesus monkey carcinoembryonic antigenand a rhesus monkey codon-optimized carcinoembryonic antigen,respectively.

The term “mammalian” refers to any mammal, including a human being.

The abbreviation “Ag” refers to an antigen.

The abbreviations “Ab” and “mAb” refer to an antibody and a monoclonalantibody, respectively.

The abbreviation “ORF” refers to the open reading frame of a gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) shows the nucleotide sequence of a codon optimized rhesusmonkey CEA cDNA (SEQ ID NO:1). See EXAMPLE 1. Panel (B) shows thepredicted amino acid sequences of rhesus monkey CEA protein deduced fromwild-type CEA nucleotide sequences isolated from two different rhesusmonkeys (SEQ ID NOs:2 and 3). Differences between the two rhesus monkeyamino acid sequences are indicated.

FIG. 2 shows a comparison between wild-type rhesus CEA expression andcodon-optimized rhesus CEA expression, as determined by Western blotanalysis. HeLa cells were either transfected with pV1J vector orinfected with Ad5 expressing rhCEA or rhCEAopt at the indicated doses.Rhesus CEA was detected as a 180-200 KDa band.

FIG. 3 shows a comparison of rhCEA and rhCEAopt expression in C57BL/6mice by ELISA. Mice were injected intramuscularly with Ad5 vectors atthe indicated doses. Expression of circulating rhCEA in Ad5 injectedmice was detected by ELISA 3 days later. Symbols represent OD₄₀₅ valuesfor each single mouse of the group. The filled circles represent thegeometric mean of each group.

FIG. 4 shows a comparison of the cellular immune response in C57BL/6mice vaccinated with rhCEA or rhCEAopt-expressing vectors. Mice wereimmunized once with DNA or Ad5 at the indicated doses. Two weeks later,an ELISPOT assay was performed to measure the cell mediated immuneresponse.

FIG. 5 depicts the humoral immune response in CEA transgenic (CEA.Tg)mice after four DNA injections. CEA.Tg mice were immunized 4 times withthe indicated plasmid DNA, expressing either human or rhesus CEA. Thetwo groups on the far right were immunized either with a 50% mix ofrhesus and human CEAopt vectors or 3 times with rhCEAopt and lastly withhCEAopt. Total IgG and IgG isotypes were measured by ELISA.

FIG. 6 depicts the humoral immune response in CEA.Tg mice after fourinjections with the indicated DNA and one Ad5 injection. IgG titer wasmeasured before (day 41) and after (day 57) Ad5 boosting. Total IgG andIgG isotypes were measured by ELISA.

FIG. 7 shows the cellular immune response in CEA.Tg mice immunized byDNA-Ad5 mixed modality. Immunizations were performed with the indicatedcombinations. Cellular immune response of groups of mice comprising 3 to4 mice/group was determined by ELISPOT assay using peptide pools A, B,C, and D as stimulators. CD8 peptide is contained in pool D.

FIG. 8 shows that different epitopes of rhesus CEA are able to elicit acellular immune response upon CEA.Tg mice immunization. The first columnlists specific CEA peptides used. Results are reported as the number ofspot forming colonies (SFC) per 10⁶ cells. Significant numbers of SFCmeasured by ELISPOT are indicated in bold. CD4+ and CD8+ epitopes, asdetermined by intracellular staining, are shown in solid gray andhatched gray cells, respectively. Epitopes that are able to activateboth CD4+ and CD8+ IFNγ secretion are also indicated in dotted cells.

FIG. 9 shows expression of rhCEA in HeLa cells infected with Ad5 or Ad24expressing rhCEAopt at the indicated doses. Rhesus CEA was detected as a180-200 KDa band.

FIG. 10 shows a comparison of expression of rhCEAopt and rhCEA in CEA.Tgmice immunized with the indicated Ad vectors. Mice were injectedintramuscularly with Ad5, Ad6 and Ad24 vectors at the dose of 1×10¹⁰ pp.Expression of circulating rhCEA was detected by ELISA 3 days after theinjection. Each symbol represents expression data from a single mouse.

FIG. 11 shows the cellular immune response in CEA.Tg mice injected withvarious prime/boost modalities. Mice were injected twice at 1×10¹⁰ pp ofadenoviral vectors with the indicated modality. The resulting immuneresponse was measured by intracellular staining (ICS) on PBMC. Resultsfor different peptide pools are expressed as a percentage of IFNγ+cells.

FIG. 12 shows results of rhesus monkey immunization protocol CV-1, inwhich Ad24rhCEAopt was used to boost the immune response elicited by DNAand Ad5-rhCEA. Panel (A) depicts the specific immunization schedule.Panels (B and C) show the immune response for the indicated peptidepools for two different rhesus monkeys, measured by ELISPOT assay.

FIG. 13 shows the results of rhesus monkey immunization protocol CV-2,in which Ad5 and Ad24-rhCEAopt were used to boost the immune responseelicited by hCEAopt expressing DNA. Panel (A) depicts the specificimmunization schedule. Panels (B and C) show the immune response for theindicated peptide pool for two different rhesus monkeys, measured byELISPOT assay.

FIG. 14 shows the results of rhesus monkey immunization protocol CV-3,in which CV33-rhCEAopt was used to boost the immune response elicited byAd5-rhCEAopt. Panel (A) depicts the specific immunization schedule.Panel (B) shows the immune response for the indicated peptide pools fortwo different rhesus monkeys, measured by ELISPOT assay.

DETAILED DESCRIPTION OF THE INVENTION

Carcinoembryonic antigen (CEA) is commonly associated with thedevelopment of adenocarcinomas. The present invention relates tocompositions and methods to elicit or enhance immunity to the proteinproduct expressed by the CEA tumor-associated antigen, wherein aberrantCEA expression is associated with the carcinoma or its development.Association of aberrant CEA expression with a carcinoma does not requirethat the CEA protein be expressed in tumor tissue at all timepoints ofits development, as abnormal CEA expression may be present at tumorinitiation and not be detectable late into tumor progression orvice-versa.

To this end, synthetic DNA molecules encoding the rhesus monkey CEAprotein are provided. The codons of the synthetic molecules are designedso as to use the codons preferred by the projected host cell, which inpreferred embodiments is a human cell. The synthetic molecules may beused for the development of recombinant adenovirus or plasmid-basedvaccines, which provide effective immunoprophylaxis againstCEA-associated cancer through neutralizing antibody and cell-mediatedimmunity. The synthetic molecules may be used as an immunogeniccomposition. This invention provides polynucleotides which, whendirectly introduced into a vertebrate in vivo, including mammals such asprimates and humans, induce the expression of encoded proteins withinthe animal.

The present invention provides synthetic DNA molecules encoding therhesus monkey CEA protein. The synthetic molecules of the presentinvention comprise a sequence of nucleotides, wherein some of thenucleotides have been altered so as to use the codons preferred by ahuman cell, thus allowing for high-level expression of rhCEA in a humanhost cell. The synthetic molecules may be used in a cancer vaccine toprovide effective immunoprophylaxis against CEA-associated carcinomasthrough neutralizing antibody and cell-mediated immunity, or as a sourceof rhesus CEA protein.

A “triplet” codon of four possible nucleotide bases can exist in over 60variant forms. Because these codons provide the message for only 20different amino acids (as well as transcription initiation andtermination), some amino acids can be coded for by more than one codon,a phenomenon known as codon redundancy. For reasons not completelyunderstood, alternative codons are not uniformly present in theendogenous DNA of differing types of cells. Indeed, there appears toexist a variable natural hierarchy or “preference” for certain codons incertain types of cells. As one example, the amino acid leucine isspecified by any of six DNA codons including CTA, CTC, CTG, CTT, TTA,and TTG. Exhaustive analysis of genome codon frequencies formicroorganisms has revealed endogenous DNA of E. coli most commonlycontains the CTG leucine-specifying codon, while the DNA of yeasts andslime molds most commonly includes a TTA leucine-specifying codon. Inview of this hierarchy, it is generally believed that the likelihood ofobtaining high levels of expression of a leucine-rich polypeptide by anE. coli host will depend to some extent on the frequency of codon use.For example, it is likely that a gene rich in TTA codons will be poorlyexpressed in E. colo, whereas a CTG rich gene will probably be highlyexpressed in this host. Similarly, a preferred codon for expression of aleucine-rich polypeptide in yeast host cells would be TTA.

The implications of codon preference phenomena on recombinant DNAtechniques are manifest, and the phenomenon may serve to explain manyprior failures to achieve high expression levels of exogenous genes insuccessfully transformed host organisms—a less “preferred” codon may berepeatedly present in the inserted gene and the host cell machinery forexpression may not operate as efficiently. This phenomenon suggests thatsynthetic genes which have been designed to include a projected hostcell's preferred codons provide an optimal form of foreign geneticmaterial for practice of recombinant DNA techniques. Thus, one aspect ofthis invention is a human CEA gene that is codon-optimized forexpression in a human cell. In a preferred embodiment of this invention,it has been found that the use of alternative codons encoding the sameprotein sequence may remove the constraints on expression of exogenousCEA protein in human cells.

In accordance with this invention, the rhesus monkey CEA gene sequencewas converted to a polynucleotide sequence having an identicaltranslated sequence but with alternative codon usage as described byLathe, “Synthetic Oligonucleotide Probes Deduced from Amino AcidSequence Data: Theoretical and Practical Considerations” J. Molec. Biol.183:1-12 (1985), which is hereby incorporated by reference. Themethodology generally consists of identifying codons in the wild-typesequence that are not commonly associated with highly expressed humangenes and replacing them with optimal codons for high expression inhuman cells. The new gene sequence is then inspected for undesiredsequences generated by these codon replacements (e.g., “ATTTA”sequences, inadvertent creation of intron splice recognition sites,unwanted restriction enzyme sites, high GC content, etc.). Undesirablesequences are eliminated by substitution of the existing codons withdifferent codons coding for the same amino acid. The synthetic genesegments are then tested for improved expression.

The methods described above were used to create synthetic gene sequencesfor rhesus monkey CEA, resulting in a gene comprising codons optimizedfor high level expression in a human cell. While the above procedureprovides a summary of our methodology for designing codon-optimizedgenes for use in cancer vaccines, it is understood by one skilled in theart that similar vaccine efficacy or increased expression of genes maybe achieved by minor variations in the procedure or by minor variationsin the sequence. One of skill in the art will also recognize thatadditional DNA molecules may be constructed that provide for high levelsof rhesus monkey CEA expression in human cells, wherein only a portionof the codons of the DNA molecules are codon-optimized.

Accordingly, the present invention relates to a synthetic polynucleotidecomprising a sequence of nucleotides encoding a rhesus monkey CEAprotein (SEQ ID NOs:2 or 3), or a biologically active fragment or mutantform of a rhesus monkey CEA protein, the polynucleotide sequencecomprising codons optimized for expression in a human host. Said mutantforms of the rhCEA protein include, but are not limited to: conservativeamino acid substitutions, amino-terminal truncations, carboxy-terminaltruncations, deletions, or additions. Any such biologically activefragment and/or mutant will encode either a protein or protein fragmentwhich at least substantially mimics the immunological properties of therhCEA protein as set forth in SEQ ID NO:2 and SEQ ID NO:3. The syntheticpolynucleotides of the present invention encode mRNA molecules thatexpress a functional rhesus monkey CEA protein so as to be useful in thedevelopment of a therapeutic or prophylactic cancer vaccine.

The present invention relates to a synthetic nucleic acid molecule(polynucleotide) comprising a sequence of nucleotides which encodes mRNAthat expresses a novel rhCEA protein as set forth in SEQ ID NO:2 and SEQID NO:3, wherein the synthetic nucleic acid molecule is codon-optimizedfor high-level expression in a human host cell. The nucleic acidmolecules of the present invention are substantially free from othernucleic acids.

The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which containthe nucleic acid molecules disclosed throughout this specification. Thesynthetic DNA molecules, associated vectors, and hosts of the presentinvention are useful for the development of a cancer vaccine.

A preferred DNA molecule of the present invention comprises thenucleotide sequence disclosed herein as SEQ ID NO:1, shown in FIG. 1(A),which encodes the rhesus monkey CEA protein shown in FIG. 1(B) (top) andset forth as SEQ ID NO:2.

The present invention also includes biologically active fraginents ormutants-of SEQ ID NO: 1, which encode mRNA expressing rhesus monkey CEAproteins. Any such biologically active fragment and/or mutant willencode either a protein or protein fragment which at least substantiallymimics the pharmacological properties of the rhCEA protein, includingbut not limited to the rhCEA proteins as set forth in SEQ ID NO:2 andSEQ ID NO:3. Any such polynucleotide includes but is not necessarilylimited to: nucleotide substitutions, deletions, additions,amino-terminal truncations and carboxy-terminal truncations. Themutations of the present invention encode mRNA molecules that express afunctional rhCEA protein in a eukaryotic cell so as to be useful incancer vaccine development.

This invention also relates to synthetic codon-optimized DNA moleculesthat encode the rhCEA protein wherein the nucleotide sequence of thesynthetic DNA differs significantly from the nucleotide sequence of SEQID NO:1, but still encodes the rhCEA protein as set forth in SEQ ID NO:2or the rhCEA protein set forth in SEQ ID NO:3. Also included within thescope of this invention are mutations in the DNA sequence that do notsubstantially alter the ultimate physical properties of the expressedprotein. For example, substitution of valine for leucine, arginine forlysine, or asparagine for glutamine may not cause a change in thefunctionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide that has properties that are different than thoseof the naturally occurring peptide. Methods of altering the DNAsequences include but are not limited to site directed mutagenesis.Examples of altered properties include but are not limited to changes inthe affinity of an enzyme for a substrate or receptor for a ligand.

The present invention also relates to rhCEAopt fusion constructs,including but not limited to fusion constructs which express a portionof the rhesus monkey CEA protein linked to various markers, includingbut in no way limited to GFP (Green fluorescent protein), the MYCepitope, GST, and Fc. Any such fusion construct may be expressed in thecell line of interest and used to screen for modulators of the rhesusmonkey CEA protein disclosed herein. Also contemplated are fusionconstructs that are constructed to enhance the immune response to rhesusmonkey CEA including, but not limited to: DOM and hsp70, and LTB.

The present invention further relates to recombinant vectors thatcomprise the synthetic nucleic acid molecules disclosed throughout thisspecification. These vectors may be comprised of DNA or RNA. For mostcloning purposes, DNA vectors are preferred. Typical vectors includeplasmids, modified viruses, baculovirus, bacteriophage, cosmids, yeastartificial chromosomes, and other forms of episomal or integrated DNAthat can encode a rhCEA protein. It is well within the purview of theskilled artisan to determine an appropriate vector for a particular genetransfer or other use.

An expression vector containing codon-optimized DNA encoding a rhCEAprotein may be used for high-level expression of rhCEA in a recombinanthost cell. Expression vectors may include, but are not limited to,cloning vectors, modified cloning vectors, specifically designedplasmids or viruses. Also, a variety of bacterial expression vectors maybe used to express recombinant rhCEA in bacterial cells if desired. Inaddition, a variety of fungal cell expression vectors may be used toexpress recombinant rhCEA in fungal cells. Further, a variety of insectcell expression vectors may be used to express recombinant protein ininsect cells.

The present invention also relates to host cells transformed ortransfected with vectors comprising the nucleic acid molecules of thepresent invention. Depending on the host cell of choice, the nucleotidesequence may be altered to include codons preferred by said host forhigh-level gene expression. Recombinant host cells may be prokaryotic oreukaryotic, including but not limited to, bacteria such as E. coli,fungal cells such as yeast, mammalian cells including, but not limitedto, cell lines of bovine, porcine, monkey and rodent origin; and insectcells including but not limited to Drosophila and silkworm derived celllines. Such recombinant host cells can be cultured under suitableconditions to produce rhCEA or a biologically equivalent form. In apreferred embodiment of the present invention, the host cell is human.As defined herein, the term “host cell” is not intended to include ahost cell in the body of a transgenic human being, human fetus, or humanembryo.

As noted above, an expression vector containing DNA encoding a rhCEAprotein may be used for expression of rhCEA in a recombinant host cell.Therefore, another aspect of this invention is a process for expressinga rhesus monkey CEA protein in a recombinant host cell, comprising: (a)introducing a vector comprising a nucleic acid that encodes rhesusmonkey CEA protein into a suitable human host cell, wherein the rhesusmonkey CEA protein comprises a sequence of amino acids as set forth inSEQ ID NO:2 or SEQ ID NO:3, and wherein the nucleic acid iscodon-optimized for optimal expression in the host cell; and, (b)culturing the host cell under conditions which allow expression of saidrhesus monkey CEA protein.

In a preferred embodiment of this aspect of the invention, the nucleicacid comprises a sequence of nucleotides as set forth in SEQ ID NO:1.

Following expression of rhCEA in a host cell, rhCEA protein may berecovered to provide rhCEA protein in active form. Several rhCEA proteinpurification procedures are available and suitable for use. RecombinantrhCEA protein may be purified from cell lysates and extracts by variouscombinations of, or individual application of salt fractionation, ionexchange chromatography, size exclusion chromatography, hydroxylapatiteadsorption chromatography and hydrophobic interaction chromatography. Inaddition, recombinant rhCEA protein can be separated from other cellularproteins by use of an immunoaffinity column made with monoclonal orpolyclonal antibodies specific for full-length rhCEA protein, orpolypeptide fragments of rhCEA protein.

The nucleic acids of the present invention may be assembled into anexpression cassette which comprises sequences designed to provide forefficient expression of the protein in a human cell. The cassettepreferably contains a full-length codon-optimized rhCEA gene, withrelated transcriptional and translations control sequences operativelylinked to it, such as a promoter, and termination sequences. In apreferred embodiment, the promoter is the cytomegalovirus promoterwithout the intron A sequence (CMV), although those skilled in the artwill recognize that any of a number of other known promoters such as thestrong immunoglobulin, or other eukaryotic gene promoters may be used. Apreferred transcriptional terminator is the bovine growth hormoneterminator, although other known transcriptional terminators may also beused. The combination of CMV-BGH terminator is particularly preferred.

In accordance with this invention, the rhCEAopt expression cassette isinserted into a vector. The vector is preferably an adenoviral vector,although linear DNA linked to a promoter, or other vectors, such asadeno-associated virus or a modified vaccinia virus, retroviral orlentiviral vector may also be used.

If the vector chosen is an adenovirus, it is preferred that the vectorbe a first-generation adenoviral vector. These adenoviral vectors arecharacterized by having a non-functional E1 gene region, and preferablya deleted adenoviral E1 gene region. In some embodiments, the expressioncassette is inserted in the position where the adenoviral E1 gene isnormally located. In addition, these vectors optionally have anon-functional or deleted E3 region. It is preferred that the adenovirusgenome used be deleted of both the E1 and E3 regions (ΔE1ΔE3). Theadenoviruses can be multiplied in known cell lines which express theviral E1 gene, such as 293 cells, or PERC.6 cells, or in cell linesderived from 293 or PERC.6 cell which are transiently or stablilytransforrned to express an extra protein. For examples, when usingconstructs that have a controlled gene expression, such as atetracycline regulatable promoter system, the cell line may expresscomponents involved in the regulatory system. One example of such a cellline is T-Rex-293; others are known in the art.

For convenience in manipulating the adenoviral vector, the adenovirusmay be in a shuttle plasmid form. This invention is also directed to ashuttle plasmid vector which comprises a plasmid portion and anadenovirus portion, the adenovirus portion comprising an adenoviralgenome which has a deleted E1 and optional E3 deletion, and has aninserted expression cassette comprising codon-optimized rhesus monkeyCEA. In preferred embodiments, there is a restriction site flanking theadenoviral portion of the plasmid so that the adenoviral vector caneasily be removed. The shuttle plasmid may be replicated in prokaryoticcells or eukaryotic cells.

In a preferred embodiment of the invention, the expression cassette isinserted into the pMRKAd5-HV0 adenovirus plasmid (See Emini et al., WO02/22080, which is hereby incorporated by reference). This plasmidcomprises an Ad5 adenoviral genome deleted of the E1 and E3 regions. Thedesign of the pMRKAd5-HV0 plasmid was improved over prior adenovectorsby extending the 5′ cis-acting packaging region further into the E1 geneto incorporate elements found to be important in optimizing viralpackaging, resulting in enhanced virus amplification. Advantageously,this enhanced adenoviral vector is capable of maintaining geneticstability following high passage propagation.

Standard techniques of molecular biology for preparing and purifying DNAconstructs enable the preparation of the adenoviruses, shuttle plasmids,and DNA immunogens of this invention.

It has been determined in accordance with the present invention that thesynthetic cDNA molecule described herein (SEQ ID NO:1), which iscodon-optimized for high-level expression in a human cell, is expressedwith greater efficiency than the corresponding wild type sequence. In aCEA transgenic mouse model, xenogeneic immunization with the codonoptimized cDNA of rhCEA breaks tolerance to rhCEA more efficiently thanthe wild type sequence. Additionally, it was shown herein that rhCEAoptis more immunogenic that rhCEA and is more efficient in eliciting bothcellular and humoral immune responses. Further, rhCEAopt expressingvectors efficiently break the immune tolerance to rhCEA in rhesusmonkeys, thus confirming the observations in mice.

Therefore, the vectors described above may be used in immunogeniccompositions and vaccines for preventing the development ofadenocarcinomas associated with aberrant CEA expression and/or fortreating existing cancers. The vectors of the present invention allowfor vaccine development and commercialization by eliminatingdifficulties with obtaining high expression levels of exogenous CEA insuccessfully transformed host organisms. To this end, one aspect of theinstant invention is a method of preventing or treating cancercomprising administering to a mammal a vaccine vector comprising asynthetic codon-optimized nucleic acid molecule, the syntheticcodon-optimized nucleic acid molecule comprising a sequence ofnucleotides that encodes a rhesus monkey CEA protein as set forth in SEQID NO:2 or SEQ ID NO:3.

In accordance with the method described above, the vaccine vector may beadministered for the treatment or prevention of cancer in any mammal. Ina preferred embodiment of the invention, the mammal is a human.

Further, one of skill in the art may choose any type of vector for usein the treatment and prevention method described. Preferably, the vectoris an adenovirus vector or a plasmid vector. In a preferred embodimentof the invention, the vector is an adenoviral vector comprising anadenoviral genome with a deletion in the adenovirus E1 region, and aninsert in the adenovirus E1 region, wherein the insert comprises anexpression cassette comprising: (a) a synthetic codon-optimizedpolynucleotide encoding a rhesus monkey CEA protein; and (b) a promoteroperably linked to the polynucleotide.

The instant invention further relates to an adenovirus vaccine vectorcomprising an adenoviral genome with a deletion in the E1 region, and aninsert in the E1 region, wherein the insert comprises an expressioncassette comprising: (a) a synthetic codon-optimized polynucleotideencoding a rhesus monkey CEA protein, wherein the polynucleotide iscodon-optimized for high expression in a human cell; and (b) a promoteroperably linked to the polynucleotide.

In a preferred embodiment of this aspect of the invention, theadenovirus vector is an Ad 5 vector.

In another preferred embodiment of the invention, the adenovirus vectoris an Ad 6 vector.

In yet another preferred embodiment, the adenovirus vector is an Ad 24vector.

In another aspect, the invention relates to a vaccine plasmid comprisinga plasmid portion and an expression cassette portion, the expressioncassette portion comprising: (a) a synthetic codon-optimizedpolynucleotide encoding a rhesus monkey CEA protein, wherein thepolynucleotide is codon-optimized for high level expression in a humancell; and (b) a promoter operably linked to the polynucleotide.

In some embodiments of this invention, the recombinant adenovirusvaccines disclosed herein are used in various prime/boost combinationswith a plasmid-based polynucleotide vaccine in order to induce anenhanced immune response. In this case, the two vectors are administeredin a “prime and boost” regimen. For example the first type of vector isadministered, then after a predetermined amount of time, for example, 2weeks, 1 month, 2 months, six months, or other appropriate interval, asecond type of vector is administered. Preferably the vectors carryexpression cassettes encoding the same polynucleotide or combination ofpolynucleotides. In the embodiment where a plasmid DNA is also used, itis preferred that the vector contain one or more promoters recognized bymammalian or insect cells. In a preferred embodiment, the plasmid wouldcontain a strong promoter such as, but not limited to, the CMV promoter.The synthetic rhesus monkey CEA gene or other gene to be expressed wouldbe linked to such a promoter. An example of such a plasmid would be themammalian expression plasmid V1Jns as described (J. Shiver et. al. inDNA Vaccines, M. Liu et al. eds., N.Y. Acad. Sci., N.Y., 772:198-208(1996), which is herein incorporated by reference).

As stated above, an adenoviral vector vaccine and a plasmid vaccine maybe administered to a vertebrate as part of a single therapeutic regimeto induce an immune response. To this end, the present invention relatesto a method of protecting a reammal from cancer comprising: (a)introducing into the mammal a first vector comprising: i) a syntheticcodon-optimized polynucleotide encoding a rhesus monkey CEA protein; andii) a promoter operably linked to the polynucleotide; (b) allowing apredetermined amount of time to pass; and (c) introducing into themammal a second vector comprising: i) a synthetic codon-optimizedpolynucleotide encoding a rhesus monkey CEA protein; and ii) a promoteroperably linked to the polynucleotide.

In one embodiment of the method of protection described above, the firstvector is a plasmid and the second vector is an adenovirus vector. In analternative embodiment, the first vector is an adenovirus vector and thesecond vector is a plasmid. In yet another embodiment, both the firstand the second vectors are adenovirus vectors.

The instant invention further relates to a method of treating a mammalsuffering from an adenocarcinoma comprising: (a) introducing into themammal a first vector comprising: i) a synthetic codon-optimizedpolynucleotide encoding a rhesus monkey CEA protein; and ii) a promoteroperably linked to the polynucleotide; (b) allowing a predeterminedamount of time to pass; and (c) introducing into the mammal a secondvector comprising: i) a synthetic codon-optimized polynucleotideencoding a rhesus monkey CEA protein; and ii) a promoter operably linkedto the polynucleotide.

In one embodiment of the method of treatment described above, the firstvector is a plasmid and the second vector is an adenovirus vector. In analternative embodiment, the first vector is an adenovirus vector and thesecond vector is a plasmid. In yet another embodiment, both the firstand the second vectors are adenovirus vectors.

The amount of expressible DNA or transcribed RNA to be introduced into avaccine recipient will depend partially on the strength of the promotersused and on the immunogenicity of the expressed gene product. Ingeneral, an immunologically or prophylactically effective dose of about1 ng to 100 mg, and preferably about 10 μg to 300 μg of a plasmidvaccine vector is administered directly into muscle tissue. An effectivedose for recombinant adenovirus is approximately 10⁶-10¹² particles andpreferably about 10⁷-10¹¹ particles. Subcutaneous injection,intradermnal introduction, impression though the skin, and other modesof administration such as intraperitoneal, intravenous, or inhalationdelivery are also contemplated. It is also contemplated that boostervaccinations may be provided. Parenteral administration, such asintravenous, intramuscular, subcutaneous or other means ofadministration with adjuvants such as interleukin 12 protein,concurrently with or subsequent to parenteral introduction of thevaccine of this invention is also advantageous.

The vaccine vectors of this invention may be naked, i.e., unassociatedwith any proteins, adjuvants or other agents which impact on therecipient's immune system. In this case, it is desirable for the vaccinevectors to be in a physiologically acceptable solution, such as, but notlimited to, sterile saline or sterile buffered saline. Alternatively, itmay be advantageous to administer an immunostimulant, such as anadjuvant, cytokine, protein, or other carrier with the vaccines orimmunogenic compositions of the present invention. Therefore, thisinvention includes the use of such imrnunostimulants in conjunction withthe compositions and methods of the present invention. Aninununostimulant, as used herein, refers to essentially any substancethat enhances or potentiates an immune response (antibody and/orcell-mediated) to an exogenous antigen. Said immunostimulants can beadministered in the form of DNA or protein. Any of a variety ofimmunostimulants may be employed in conjunction with the vaccines andimmunogenic compositions of the present inventions, including, but notlimited to: GM-CSF, IFNα, tetanus toxoid, IL12, B7.1, LFA-3 and ICAM-1.Said immunostimulants are well-known in the art. Agents which assist inthe cellular uptake of DNA, such as, but not limited to calcium ion, mayalso be used. These agents are generally referred to as transfectionfacilitating reagents and pharmaceutically acceptable carriers. Those ofskill in the art will be able to determine the particularimmunostimulant or pharmaceutically acceptable carrier as well as theappropriate time and mode of administration.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to those precise embodiments, and that various changesand modifications may be effected therein by one skilled in the artwithout departing from the scope or spirit of the invention as defmed inthe appended claims.

The following examples illustrate, but do not limit the invention.

EXAMPLE 1

Construction of Codon-Optimized rhCEA

Wild-type rhesus monkey amino acid sequences were deduced fromnucleotide sequences isolated from two different rhesus monkeys. Toisolate and determine wild-type nucleotide sequences encoding the rhesusmonkey CEA protein, nucleotide sequences from the 5′ and 3′ untranslatedregions (UTR) of all known members of the human CEA family were alignedto identify highly conserved regions of the CEA DNA. Based on the CEAgene family homologies, degenerate oligonucleotide primers were designedand PCR conditions were optimized to amplify the rhesus CEA cDNA byreverse transcriptase polymerase chain reaction using RNA isolated fromcolon samples from two different Rhesus monkeys (Macaca Mulatta).Amplified PCR products of about 2100 bp, the expected size for aCEACAM-5 homolog, were independently obtained from both RNA samples andwere purified from agarose gel. Partial sequence analysis of both PCRproducts revealed high homology with human CEACAM-5. The entire genesequence was obtained by purifying DNA fragments. Comparison of therhCEA nucleotide sequences obtained from two rhesus monkeys indicatedthat there were two nucleotide differences, which code for two differentproteins. The predicted protein sequences are shown in FIG. 1(B) (SEQ IDNOs:2 and 3).

Based on the predicted amino acid sequence of one of the two rhCEAproteins described (SEQ ID NO:2), a rhesus CEA cDNA was designed thatcomprises optimal codons for high expression in human cells, using theVector NTI program algorithm (InforMax, Rockville, Md.). To increase thelevel of transcription, an optimized Kozak sequence was inserted 5′ tothe ATG. Moreover, two consecutive stop codons were inserted downstreamof the coding sequence. The gene was synthesized by Bionexus, Inc(Oakland, Calif.) by PCR-mediated oligonucleotide assembly and cloned inthe vector pCR-blunt (Invitrogen, Carlsbad, Calif.). To verify thesequence, both strands of the gene were sequenced by using ABI 377automated sequencer. Autoassembler program was used to compare thesequence data of the synthesized gene with the expected sequence. Theopen reading frame of the optimized rhesus CEA sequence is depicted inFIG. 1(A) (SEQ ID NO:1).

EXAMPLE 2

Plasmid Constructs and Adenovirus Generation

pV1J-rhCEAopt: RhCEAopt was excised as an EcoRI fragment frompCR-blunt-rhCEAopt vector and inserted in pV1J-nsA vector, obtainingpV1J-RhCEAopt.

pAd5-rhCEAopt and pAd24-rhCEAopt: For adenovirus 5 generation,pMRK-rhCEAopt was obtained by subcloning rhCEAopt as a HincII/XhoIfragment in SwaI/SalI sites of the polyMRK vector (See Emini et al., WO02/22080, which is hereby incorporated by reference). For Ad24generation, the expression cassette was excised from pMRK-rhCEAopt as anSspI/AscI fragment and inserted in the shuttle vector pABS-Ad17-3 in theEcoRI site, thus generating pABS-Ad17-rhCEAopt. A PacI/StuI fragmentfrom pMRK-rhCEAopt and a XhoI/XbaI fragment from pABS-Ad17-rhCEAoptcontaining the expression cassette for rhCEAopt and E1 flanking Ad5 andAd17/24 regions respectively, were recombined to ClaI linearized pAd5 orSwaI linearized pAd24 using BJ5183 E. Coli cells. The resulting plasmidswere pAd5-rhCEAopt and pAd24-rhCEAopt. These plasmids were cut with PacIor PmeI respectively, to release the adenovirus ITRs and transfected inPerC-6 cells by Lipofectamine 2000 (Life Technologies, Carlsbad,Calif.). The amplification of the vectors was carried out through serialpassages. Ad5-rhCEAopt and Ad24rhCEAopt were purified through a standardCsCl purification protocol and extensively dialyzed against A105 buffer(5 mM Tris pH 8.0, 1 mM MgCl₂, 75 mM NaCl, 5% Sucrose, 0.005% Tween20).

pV1J-rhCEA. Ad5-rhCEA and Ad6-rhCEA: RhCEA was excised with PstI/XhoIfrom pCMV-script EX phagemid vector and inserted in pBluescript H KSvector, obtaining pBS-RhCEA. The insert was entirely sequenced and thensubcloned as SmaI/XhoI fragment in pV1JnsA vector, obtaining pV1J-RhCEA.The shuttle plasmid pMRK-RhCEA for adenovirus generation was obtained bysubeloning the same fragment in the polyMRK vector. A PacI/StuI fragmentfrom pMRK-RhCEA containing the expression cassette for RhCEA and E1flanking Ad5 regions was recombined to ClaI linearized pAd5 or pAd6 inBJ5183 E. Coli cells. The resulting plasmids were pAd5-RhCEA andpAd6-RhCEA. Both plasmids were cut with PacI to release the adenovirusITRs and transfected in PerC-6 cells. Viral amplification was carriedout through serial passages. Ad5-RhCEA and Ad6-RhCEA were purified usinga standard CsCl purification protocol and extensively dyalized againstA105 buffer (5 mM Tris pH 8.0, 1 mM MgC12, 75mM NaCl, 5% sucrose, 0.005%Tween20).

EXAMPLE 3

In vitro Expression of Rhesus CEA

Rhesus CEA expression from the constructs described above was verifiedby western blot and FACS analysis. Plasmids were transfected in HeLacells with Lipofectamine 2000 (Life Technologies) according tomanufacturer directions. Adenovirus infections were performed inserum-free medium for 30 min at 37° C., then fresh medium was added tocells. 48 hours later, whole cell lysates were analyzed by western blotusing a rabbit polyclonal serum against human CEA (Fitzgerald IndustriesInternational Inc., Concord Mass., 1:1500 dilution). Rhesus CEA wasdetected as a 180-200KDa band.

Western blot analysis demonstrated that transfection of HeLa cells withan expression plasmid (pV1J-rhCEAopt) carrying the optimized rhesus CEAcDNA (rhCEAopt) at different doses showed 100 fold greater proteinlevels than a similar vector carrying the native cDNA (pV1J-rhCEAopt).See FIG. 2. Similarly, infection of HeLa cells with a first generationΔE1-ΔE3 adenovirus vector expressing rhCEAopt (MRKAd5-rhCEAopt) showed asignificant improvement in expression level (FIG. 2). Thus, theoptimization of the rhesus CEA coding sequence effectively enhanced thelevel of expression of rhCEA in vitro.

EXAMPLE 4

Mice Immunization

C57BL/6 mice (H-2^(b)) were purchased by Charles River (Lecco, Italy).CEA transgenic (CEA.tg) mice (H-2^(b)) were provided by J. Prirnus(Vanderbilt University) and kept in standard conditions.

For electro gene transfer (EGT), mice quadriceps were either surgicallyexposed or directly injected with the indicated doses of pV1J-rhCEA orpV1J-rhCEAopt and electrically stimulated as previously described(Rizzuto at al. Proc. Natl. Acad. Sci U.S.A. 96(11): 6417-22 (1999)).

For adenovirus injection, Ad5-rhCEA, Ad6-rhCEA, Ad5-rhCEAopt orAd24-rhCEAopt were injected in mice quadriceps at the indicated doses.

EXAMPLE 5

Rhesus CEA Ouantitation and Antibody Titration

Sera for antibody titration were obtained by retro-orbital bleeding. Forrhesus CEA measurement in the blood, Elisa plates (Nunc maxisorp) werecoated O/N at 4° C. with a polyclonal anti rhCEA mouse serum incarbonate buffer (50 mM NaHCO₃ pH 9.4). Plates were then blocked withPBS containing 5% BSA for 1 hr at 37° C. Cell supernatants or sera werethen diluted in PBS 5% BSA and incubated for 2 hr at RT. After washing 5times with PBS/0.05% Tween 20, anti-CEA rabbit polyclonal antibody wasadded at 1:2000 dilution and incubated for further 2 hr. Plates werethen washed again and detecting antibody anti-rabbit IgG-AP conjugatedwas added at 1:2000 dilution for 1 hr at RT. Final detection was donewith 100 μl/well p-nitrophenyl phosphate disodium, 1.0 mg/ml in 10%diethanolamine buffer, pH 9.8 containing 0.5 mm MgCl2 and reading atOD₄₀₅.

For antibody titration, Elisa plates were coated with 100 ng/well CEAprotein (Fitzgerald, highly pure CEA), diluted in coating buffer andincubated O/N at 4° C. Mouse sera were diluted in PBS 5% BSA. Pre-immunesera were used as background. Diluted sera were incubated O/N at 4° C.Washes were carried out with PBS, 1% BSA, 0.05% tween 20. Detectingantibody (goat anti-mouse IgG Peroxidase, Sigma), was diluted 1/2000 inPBS, 5%BSA and incubated for 2-3 hr at RT on a shaker. After washing,plates were developed with 100 μl/well of TMB substrate (PierceBiotechnology, Inc., Rockford, Ill.). The reaction was stopped with 25μl/well of 1M H₂SO₄ solution and plates were read at 450 nm/620 nm.Anti-CEA serum titers were calculated as the limiting dilution of serumproducing an absorbance at least 3-fold greater than the absorbance ofautologous pre-immune serum at the same dilution.

EXAMPLE 6

ELISPOT Assay for IFN-γ

96-well MAIP plates (Millipore) were coated with purified rat anti-mouseIFN-γ (IgG1, clone R4-6A2, Pharmingen) at 2.5 μg/ml in sterile PBS,aliquoted at 100 μl per well. After washing with sterile PBS, blockingof plates was done with 200 μl per well of R10 medium at 37° C. for atleast 2 hours.

For preparation of splenocytes, spleens were removed from sacrificedmice in a sterile manner and disrupted by scratching through a grid. Redblood cell osmotic lysis was obtained by adding 1 ml of 0.1 ×PBS to cellpellet and vortexing for no more than 15 s. 1 ml of 2×PBS was then addedand the volume was brought up to 4 ml with PBS 1×. After spinning at1200 rpm for 10 minutes at RT, the cell pellet was resuspended in 1 mlof R10 medium and viable cells were counted.

Splenocytes were plated at 5×10⁵ and 2.5×10⁵/well with 1 μg/ml eachpeptide in R10 and incubated for 16-20h in a CO₂ incubator at 37° C.Concanavalin A (ConA) at 5 μg/ml was used as positive internal controlfor each mouse. After washing with PBS, 0.05% Tween 20, plates wereincubated O/N at 4° C. with 50 μl/well of biotin-conjugated ratanti-mouse IFN-γ (Rat IgG1, clone XMG 1.2, PharMingen), diluted 1:250 inassay buffer (PBS-5%FBS-0.005%Tween-20). The next day, plates werewashed and incubated for 2 h at RT with streptavidin-AP conjugate(Pharmingen, San Jose, Calif.) diluted 1:2500 in assay buffer. Afterextensive washing, plates were developed by addition of 50 μl/wellNBT/B-CIP (Pierce) until development of spots was observed through themicroscope. The reaction was stopped by washing plates thoroughly withdistilled water. Plates were allowed to air-dry completely, and spotswere counted using an automated ELISPOT reader.

EXAMPLE 7

Intracellular Staining for IFN-γ

For PBMC preparation, about 200 μl blood were obtained from each mouseby retro-orbital bleeding and heparinized. Erythrocyte lysis wasobtained by incubation for 10 min with ACK lysing buffer (LifeTechnologies). After centrifugation, white cells were resuspended in R10medium. 1-2×10⁶ splenocytes or PBMC were resuspended in 1 ml R10.Antigen peptides or peptide pools were added to a final concentration of1 μg/ml with Brefeldin A.

After 12 hours incubation at 37° C., cells were washed with 3 ml FACSbuffer (PBS, 1% FCS) and centrifuged for 10 min at RT. Incubation withanti-mouse CD16/CD32 was carried out in 100 μl FACS buffer for 15 min at4° C. After washing, for the surface antigen staining, APC conjugatedanti-mouse CD3 ε, PE conjugated anti-mouse CD4, PerCP conjugatedanti-mouse CD8 α all diluted 1:50 in FACS buffer were added in 100 μlfinal volume and incubated for 30 min at RT in the dark. After washingwith PermWash (Pharmingen), cells were resuspended in 100 μl ofCytofix-Cytoperm solution (Pharmningen), vortexed and incubated for 20min at 4° C. in the dark. For intracellular staining, cells wereincubated with FITC conjugated anti-mouse interferon-γ diluted 1:50 inPermWash (100 μl final volume) for 30 min at RT in the dark Afterwashing, cells were resuspended in 250-300 μl 1% formaldehyde in PBS andanalyzed with a FACScalibur (Becton Dickinson, San Jose, Calif.).

EXAMPLE 8

Expression and Immunization Studies in Wild-Type Mice

Transfection of HeLa cells with constructs carrying wild-type orcodon-optimized rhesus CEA demonstrated that optimization of the rhesusCEA coding sequence enhanced the level of expression of rhCEA in vitro.See EXAMPLE 3. To determine whether a higher level of expression couldbe measured also in vivo, C57BL/6 mice were either injectedintramuscularly with pV1J-rhCEAopt followed by electro gene transfer(EGT) or with MRKAd5-rhCEAopt and compared with similar vectors carryingwild type CEA. Four days later, mice were bled and circulating levels ofrhCEA were measured by ELISA. Greater protein levels were obtained inAd5-rhCEAopt injected mice at different doses than those detected uponinjection of vectors encoding the wt rhCEA (FIG. 3). No significantexpression of rhesus CEA could be measured upon DNA plasmid injection(data not shown).

To verify whether the higher expression was related to a higherimmunogenicity, cellular immune response in injected mice was measuredby ELISPOT using rhCEA peptides as stimulators. Significant enhancementof the immune response could be measured using rhCEAopt expressing DNAvectors compared to rhCEA vectors (FIG. 4). These data show thatoptimization of rhCEA cDNA was effective in augmenting the level ofexpression of rhesus CEA in vivo and, more importantly, in enhancingcell mediated immune response in wild type C57BL/6 mice.

EXAMPLE 9

Mixed Modality Vaccination Studies in CEA Transgenic Mice

Immunization experiments with wild-type rhesus monkey CEA demonstratedthe ability of rhCEA xenogeneic vaccination to elicit an immune responseagainst human CEA as self-antigen. Briefly, CEA transgenic (CEA.Tg) micewere immunized with vectors encoding human (self) or rhesus CEA (xeno)and breakage of immune tolerance was achieved using rhCEA bearingvectors. CEA.Tg mice are transgenic mice that express human CEA as aself-antigen with a tissue distribution similar to that of humans.

To demonstrate if rhCEAopt expression vectors could better break theimmune tolerance to human CEA, CEA.Tg mice were immunized with DNAplasmids followed by EGT. Mice were injected 4 times at weekly intervalswith pV1J vector expressing either hCEA, hCEAopt, rhCEA or rhCEAopt.Moreover, to stimulate the immune response against the xeno-antigen andthen direct it to the self antigen to break the immune tolerance, onegroup received 4 injections of a 50% mix containing hCEAopt/rhCEAoptvectors and a second group was treated with 3 injections ofpV1J-rhCEAopt and a fourth injection of pV1J-hCEAopt.

No detectable cell-mediated immune response could be measured in anyexperimental group after 4 DNA injections as measured by ICS from PBMC(data not shown). Unlike the cellular immune response, an enhancedhumoral immune response to hCEA was detected in CEA.Tg mice vaccinatedwith rhCEAopt. Antibody measurements obtained by ELISA assaydemonstrated a 10-fold increase in the group treated with rhCEAoptcompared to the hCEAopt immunized group (FIG. 5). Consistent with thisresult, significant IgG titer (>1:1000) was obtained only when rhCEAoptwas present in the immunization schedule. The anti-CEA Ab titer in miceimmunized with wild-type rhesus CEA was not significantly different fromthe Ab titer in mice treated with hCEAopt (data not shown).

IgG isotype titer was also measured as a quality of the response. Anincreased IgG1/IgG2a ratio was obtained in mice immunized with rhCEAoptrelative to those mice vaccinated with the hCEA and hCEAopt expressingvectors, which suggests that the xeno-gene elicited an enhanced Th2-typeimmune response.

Each group of mice was then boosted with an Ad5 vector expressing thetransgene of the fourth DNA injection. Thus, mice immunized with thepV1J mix described above received a 50% mix of Ad5-rhCEAopt andAd5-hCEAopt as a booster. Similarly, mice vaccinated with 3 injectionsof rhCEAopt followed by one of pV1J-hCEAopt were boosted withAd5-hCEAopt.

Sixteen days after the booster injection, the humoral immune responsewas measured by ELISA assay. Results demonstrate that the Ad5 boostgreatly enhanced the antibody titer in rhCEAopt immunized mice (average1:150000), but not significantly in the other groups (FIG. 6). Thus, theuse of rhCEAopt as immunogen induces high cross reactive humanCEA-specific antibody response—300—fold higher than hCEA containingvectors.

To verify whether the cell mediated immune response against human CEAwas elicited in the above-described groups of mice after Ad5 boosting,an ELISPOT IFNγ assay was performed after stimulation with human CEAspecific peptides. 15mer peptides covering the entire human CEA protein,divided into four sections, were assembled, generating pools A, B, C andD. Results demonstrate that optimization of the CEA nucleotide sequenceincreased the immune response to human CEA (FIG. 7). In fact, bothhCEAopt and rhCEAopt increased the number of measured effectors.Interestingly, immunization with rhCEAopt alone or in combination withhCEAopt changed the quality and distribution of the cell mediatedresponse along the human CEA protein. Additionally, increased SFC weremeasured with pools B and C as stimulators in these groups compared tohCEAopt immunized mice.

EXAMPLE 10

Identification of Immunogenic Peptides

To identify immunogenic regions of the human and rhesus CEA proteins, anELISPOT assay was performed using individually peptides spanning the twoproteins. Responsive peptides identified by ICS in the rhesus CEAprotein were significantly different than those identified in the humanprotein (FIG. 8). Some responses were comparable or even higher againsthuman peptides than against the corresponding region of rhesus proteinwhen rhCEAopt was included in the immunization protocol.

For the human CEA protein, CD4+ (CEA44 (a.a. 173 to a.a. 187)−CEA45(a.a.177 to a.a. 191); CEA89 (a.a. 353 -a.a. 367)−CEA90 (a.a. 357 - a.a.371); and CEA 1l0( a.a. 437 to a.a. 451)) and CD8+ (CEA5(aa.17 -a.a.31))epitopes were induced by rhCEAopt or rhCEAopt/hCEAopt sequentialimmunizations. This enhanced CD4+ response compared to hCEAopt immunizedgroup could be related to the increased antibody titer measured inrhCEAopt immunized mice.

For the rhesus CEA protein, specific CD4+ responses were measured withCEA22 (a.a.85-a.a.99) and CEA110 (a.a.437-a.a.451), while CEA77(a.a.305-a.a.319), CEA121 (a.a.481-a.a.495) and CEA142 (a.a.565-a.a.579)were strong CD8+ epitopes. Interestingly, a peptide able to activateboth CD4+ and CD8+ IFNγ secretion was identified (CEA134(a.a.533-a.a.547). These results show that rhCEAopt used as xeno-antigeninduced a strong and qualitatively different cross reactive immnuneresponse to human CEA.

EXAMPLE 11

Expression and Immunization with Different Adenovirus Serotypes

To test alternative Ad serotypes as boosters of the immune response,Ad24-rhCEAopt, which comprises an Ad24 vector expressing codon-optimizedrhesus CEA (see EXAMPLE 2), was constructed as a potential booster ofthe immune response elicited in AdS injected animals. Rhesus CEAexpression was verified in vitro and compared to AdS-rhCEAopt (FIG. 9).A 50-100 fold lower protein level was obtained with Ad24 infection. Thisobservation could be due to a different ratio of physical/transducingparticles between Ad5 and Ad24 serotypes.

To verify the level of expression in vivo, CEA.Tg mice were injectedwith adeno vectors expressing either rhCEA or rhCEAopt (FIG. 10). Thesystemic expression of both AdS-rhCEAopt and Ad24-rhCEAopt wasdetectably higher than expression of Ad5 or Ad6 vectors comprisingwild-type gene. According to in vitro data, Ad24 mediated expression waslower than Ad5.

The immunogenicity of Ad24 was then tested in CEA.Tg mice. Immuneresponses elicited by the following prime- boost modalities werecompared: Ad5- Ad24, Ad24- Ad24 and Ad5-Ad5. Fourteen days after theboost, peripheral cell mediated immune response was measured by ICS.

The results indicate that the Ad5-Ad5 protocol elicited a significantlyhigher cell mediated inmnune response against different CEA peptidepools than the Ad24-Ad24 protocol (FIG. 11). Interestingly, the Ad5-Ad24protocol elicited a response comparable to Ad5-Ad5. These experimentsdemonstrate that Ad24 is a good booster of the immune response elicitedby other Ad serotypes.

EXAMPLE 12

Immunization of Rhesus Macagues with rhCEAopt: Protocol CV-1

In order to assess the efficiency of immunization of rhesus macaques(macaca mulatta) with the optimized version of rhesus CEA, immunizationstudies were performed at the Biomedical Primate Research Centre (BPRC,Rijswijk, The Netherlands). Such immunization studies were designed toevaluate both B and T cell responses to immunization with the rhesus CEAantigen.

In a first study (CV-1, FIG. 12), 1 group of monkeys (consisting of 2males and 2 females) was immunized with a plasmid DNA vector oradenovirus vector expressing the wild-type or optimized version ofrhesus CEACAM-5. For priming, animals were vaccinated intramuscularlywith plasmid DNA expressing wild-type rhCEA at weeks 0, 4, 8, 12, 16,and 24 by injection of DNA followed by electrical stimulation. The DNAinjection consisted of a I ml solution (split over 2 sites with 0.5ml/site) containing 5 mg plasmid DNA for animals weighing 2-5 kilos.Animals were injected under anesthesia (mixture of ketamine/xylazine).

For electrostimulation, 2 trains of 100 square bipolar pulses (1 seceach), were delivered every other second for a total treatment time of 3sec. The pulse length was 2 msec/phase with a pulse frequency andamplitude of 100 Hz and 100 mA (constant current mode), respectively.

To measure the immune response to CEA using the above immunizationprotocol, blood samples were collected every four weeks for a totalduration of one year. The humoral response was measured by ELISA assayand the cell mediated response was measured by IFNγ Elispot assay (FIG.12B). Since no significant immune response was obtained at week 16, twofurther injections (week 24 and 28) were carried out using Ad5expressing the wild-type version of the tumour antigen. Upon Ad5injection, the immune response against rhCEA could be measured for twomonkeys (RI137 and CO12) covering peptide pool C and pool B+C,respectively. At week 35, the immune response started to decline in bothmonkeys. To maintain the immune response, Ad24 expressing the optimizedversion of rhCEA was injected at weeks 35 and 40, resulting in ameasurable boosting effect in monkeys RI137 and CO12 at weeks 40 and 44,respectively.

These data show that adenovirus vectors are effective in inducing aspecific immune response to rhCEA in rhesus monkeys after priming withDNA. These results further indicate that Ad24-rhCEAopt is a good vaccinebooster.

EXAMPLE 13

Immunization of Rhesus Macaques with rhCEAopt: Protocol CV-2

A second series of immunization studies (CV-2, FIG. 13) was performed inorder to assess the efficiency of immunization of Rhesus macaques(macaca mulatta) with the rhesus homologues of the human tumour antigensHER2/neu, Ep-CAM and CEA, which are all expressed in colorectalcarcinomas. Protocols were designed to evaluate both B and T cellresponses to these tumor antigens in combination.

In this study, a group of 4 rhesus monkeys was immunized with a mixtureof plasmid DNA vectors expressing the human tumour-associated antigens(TAAs) Ep-CAM, CEA, and HER2/neu as xeno-antigens. For priming, animalswere vaccinated intramuscularly with injections at weeks 0, 4, 8, 12 andl6 followed by electrostimulation. The DNA injection consisted of a I mlsolution (split over 2 sites with 0.5 ml/site) containing 6 mg plasmidDNA for animals weighing 2-5 kilos. Subsequently, monkeys received twoinjections of Ad5 (weeks 27+31) followed by two injections of Ad24(weeks40+44) expressing the optimized versions of the three TAAs. Eachinjection comprised a mnixture of 1×10e11pp of Ad-rhCEAopt, 1×10e11 ofAd-rhEpCAMopt and 1×10-e11 of Ad-rhHER2opt, serotype 5 or 24.

Breakage of immune tolerance was obtained in two monkeys (RI497 andRI512, FIG. 13(B)) when Ad5 injection was used as booster and maintainedby Ad24 successive injections. To determine if this immune response willbe maintained for longer periods of time, the CV-2 protocol is followedfor additional weeks and the resulting immune response is measured everyfour weeks.

The results of the CV-2 protocol show that adenoviral vectors can beused to induce and maintain an effective immune response against rhesusCEA and, importantly, that this immune response can be achieved in thecontext of a multiple vaccination schedule.

EXAMPLE 14

Immunization of Rhesus Macaques with rhCEAopt: Protocol CV-3

To determine if an effective immune response against rhCEA can beobtained using only adenovirus vectors to prime and boost, a thirdstudy, based exclusively on adenoviral vectors was designed (ProtocolCV-3, FIG. 14). In comparison with the previous protocols, the number ofinjections for each virus increased from two to three at 0, 2 and 4weeks for Ad5. additionally, CV33 injections were given at 16, 18 and 20weeks.

Similar to the CV-2 protocol (EXAMPLE 13), 4 monkeys were immunizedsimultaneously with optimized rhCEA, rhEpCAM and rhHER2 expressingvectors. Consistent breakage of immune tolerance to rhCEA was measuredat week 4 in two monkeys (RI001 and RI373, FIG. 14(B)). This responsewas efficiently boosted by a different Ad serotype (CV33) expressing theoptimized tumor antigens.

To determine if this immune response is maintained for longer periods oftime, the CV-3 protocol is followed for additional weeks and theresulting immune response is measured every four weeks.

These data show that adenoviral vectors are sufficient to break theimmune tolerance to rhCEA and maintain the response over time in arhesus monkey animal model.

1. A synthetic nucleic acid molecule comprising a sequence ofnucleotides that encodes a rhesus monkey carcinoembryonic antigen (CEA)protein as set forth in SEQ ID NO:2 or SEQ ID NO:3, the syntheticnucleic acid molecule being codon-optimized for high level expression ina human cell. 2-5. (canceled)
 6. The synthetic nucleic acid molecule ofclaim 1 wherein the sequence of nucleotides comprises the sequence ofnucleotides set forth in SEQ ID NO:
 1. 7. A vector comprising thenucleic acid molecule of claim
 1. 8. A host cell comprising the vectorof claim
 7. 9. A process for expressing a rhesus monkey carcinoembryonicantigen (CEA) protein in a recombinant host cell, comprising: (a)introducing a vector comprising the nucleic acid of claim 1 into asuitable host cell; and, (b) culturing the host cell under conditionswhich allow expression of said rhesus monkey CEA protein.
 10. A methodof preventing or treating cancer comprising administering to a human avaccine vector comprising the synthetic nucleic acid molecule of claim1,
 11. (canceled)
 12. A method according to claim 10 wherein the vectoris an adenovirus vector or a plasmid vector. 13-14. (canceled)
 15. Anadenovirus vaccine vector comprising an adenoviral genome with adeletion in the E1 region, and an insert in the E1 region, wherein theinsert comprises an expression cassette comprising: (a) a polynucleotideencoding a rhesus monkey carcinoembrvonic antigen (CEA) protein as setforth in SEO ID NO:2 or SEQ ID NO:3 the polynucleotide beingcodon-optimized for high-level expression in a human cell; and (b) apromoter operably linked to the polynucleotide.
 16. An adenovirus vectoraccording to claim 15 wherein the adenoviral genome is selected from theg:roup consisting of: Ad5, Ad6 and Ad24. 17-18. (canceled)
 19. A vaccineplasmid comprising a plasmid portion and an expression cassette portion,the expression cassette portion comprising: (a) a polynucleotideencoding a rhesus monkey carcinoembryonic antigen (CEA) protein as setforth in SEQ ID NO:2 or SEQ ID NO:3, the polynucleotide beingcodon-optimized for high-level expression in a human cell; and (b) apromoter operably linked to the polynucleotide.
 20. A method ofprotecting a mammal from cancer comprising: (a) introducing into themammal a first vector comprising: (i) a codon-optimized polynucleotideencoding a rhesus monkey carcinoembryonic antigen (CEA) protein; and(ii) a promoter operably linked to the polynucleotide; (b) allowing apredetermined amount of time to pass; and (c) introducing into themammal a second vector comprising: (i) a codon-optimized polynucleotideencoding a rhesus monkey CEA protein; and (ii) a promoter operablylinked to the polynucleotide.
 21. A method according to claim 20 whereinthe first vector is a plasmid and the second vector is an adenovirusvector.
 22. A method according to claim 20 wherein the first vector isan adenovirus vector and the second vector is a plasmid. 23-29.(canceled)